Accurate segregation of replicated chromosomes is critical for cells to maintain genomic stability. Chromosome missegregation has been associated with the majority of human cancers. Segregation is facilitated in part by the kinetochore, a protein complex that connects the centromere of chromosomes to the microtubules of the mitotic spindle. The work described in this proposal uses Saccharomyces cerevisiae to address regulatory and assembly requirements for kinetochores on endogenous and "new" centromeres. Cell cycle requirements for kinetochore assembly will be determined by asking if kinetochore proteins can incorporate into kinetochores while the cells are held in G1, S, or M of the cell cycle. Conditional centromeres, not available in multicellular eukaryotes, will also be used here to identify epigenetic factors that may distinguish endogenous from "new" kinetochores. Finally, an epistasis analysis among kinetochore proteins will establish a dependence for assembly relationship between among the kinetochore proteins. Since many of the kinetochore proteins are conserved between S. cerevisiae and other eukaryotes, these studies can be extended to other organisms.